77 research outputs found
Metabolomics-Based Elucidation of Active Metabolic Pathways in Erythrocytes and HSC-Derived Reticulocytes
A detailed
analysis of the metabolic state of human-stem-cell-derived
erythrocytes allowed us to characterize the existence of active metabolic
pathways in younger reticulocytes and compare them to mature erythrocytes.
Using high-resolution LCāMS-based untargeted metabolomics,
we found that reticulocytes had a comparatively much richer repertoire
of metabolites, which spanned a range of metabolite classes. An untargeted
metabolomics analysis using stable-isotope-labeled glucose showed
that only glycolysis and the pentose phosphate pathway actively contributed
to the biosynthesis of metabolites in erythrocytes, and these pathways
were upregulated in reticulocytes. Most metabolite species found to
be enriched in reticulocytes were residual pools of metabolites produced
by earlier erythropoietic processes, and their systematic depletion
in mature erythrocytes aligns with the simplification process, which
is also seen at the cellular and the structural level. Our work shows
that high-resolution LCāMS-based untargeted metabolomics provides
a global coverage of the biochemical species that are present in erythrocytes.
However, the incorporation of stable isotope labeling provides a more
accurate description of the active metabolic processes that occur
in each developmental stage. To our knowledge, this is the first detailed
characterization of the active metabolic pathways of the erythroid
lineage, and it provides a rich database for understanding the physiology
of the maturation of reticulocytes into mature erythrocytes
Table_1_Synergistic Killing of Polymyxin B in Combination With the Antineoplastic Drug Mitotane Against Polymyxin-Susceptible and -Resistant Acinetobacter baumannii: A Metabolomic Study.XLSX
<p>Polymyxins are currently used as the last-resort antibiotics against multidrug-resistant Acinetobacter baumannii. As resistance to polymyxins emerges in A. baumannii with monotherapy, combination therapy is often the only remaining treatment option. A novel approach is to employ the combination of polymyxin B with non-antibiotic drugs. In the present study, we employed metabolomics to investigate the synergistic mechanism of polymyxin B in combination with the antineoplastic drug mitotane against polymyxin-susceptible and -resistant A. baumannii. The metabolomes of four A. baumannii strains were analyzed following treatment with polymyxin B, mitotane and the combination. Polymyxin B monotherapy induced significant perturbation in glycerophospholipid (GPL) metabolism and histidine degradation pathways in polymyxin-susceptible strains, and minimal perturbation in polymyxin-resistant strains. Mitotane monotherapy induced minimal perturbation in the polymyxin-susceptible strains, but caused significant perturbation in GPL metabolism, pentose phosphate pathway and histidine degradation in the LPS-deficient polymyxin-resistant strain (FADDI-AB065). The polymyxin B ā mitotane combination induced significant perturbation in all strains except the lipid A modified polymyxin-resistant FADDI-AB225 strain. For the polymyxin-susceptible strains, the combination therapy significantly perturbed GPL metabolism, pentose phosphate pathway, citric acid cycle, pyrimidine ribonucleotide biogenesis, guanine ribonucleotide biogenesis, and histidine degradation. Against FADDI-AB065, the combination significantly perturbed GPL metabolism, pentose phosphate pathway, citric acid cycle, and pyrimidine ribonucleotide biogenesis. Overall, these novel findings demonstrate that the disruption of the citric acid cycle and inhibition of nucleotide biogenesis are the key metabolic features associated with synergistic bacterial killing by the combination against polymyxin-susceptible and -resistant A. baumannii.</p
Table_3_Synergistic Killing of Polymyxin B in Combination With the Antineoplastic Drug Mitotane Against Polymyxin-Susceptible and -Resistant Acinetobacter baumannii: A Metabolomic Study.XLSX
<p>Polymyxins are currently used as the last-resort antibiotics against multidrug-resistant Acinetobacter baumannii. As resistance to polymyxins emerges in A. baumannii with monotherapy, combination therapy is often the only remaining treatment option. A novel approach is to employ the combination of polymyxin B with non-antibiotic drugs. In the present study, we employed metabolomics to investigate the synergistic mechanism of polymyxin B in combination with the antineoplastic drug mitotane against polymyxin-susceptible and -resistant A. baumannii. The metabolomes of four A. baumannii strains were analyzed following treatment with polymyxin B, mitotane and the combination. Polymyxin B monotherapy induced significant perturbation in glycerophospholipid (GPL) metabolism and histidine degradation pathways in polymyxin-susceptible strains, and minimal perturbation in polymyxin-resistant strains. Mitotane monotherapy induced minimal perturbation in the polymyxin-susceptible strains, but caused significant perturbation in GPL metabolism, pentose phosphate pathway and histidine degradation in the LPS-deficient polymyxin-resistant strain (FADDI-AB065). The polymyxin B ā mitotane combination induced significant perturbation in all strains except the lipid A modified polymyxin-resistant FADDI-AB225 strain. For the polymyxin-susceptible strains, the combination therapy significantly perturbed GPL metabolism, pentose phosphate pathway, citric acid cycle, pyrimidine ribonucleotide biogenesis, guanine ribonucleotide biogenesis, and histidine degradation. Against FADDI-AB065, the combination significantly perturbed GPL metabolism, pentose phosphate pathway, citric acid cycle, and pyrimidine ribonucleotide biogenesis. Overall, these novel findings demonstrate that the disruption of the citric acid cycle and inhibition of nucleotide biogenesis are the key metabolic features associated with synergistic bacterial killing by the combination against polymyxin-susceptible and -resistant A. baumannii.</p
Table_4_Synergistic Killing of Polymyxin B in Combination With the Antineoplastic Drug Mitotane Against Polymyxin-Susceptible and -Resistant Acinetobacter baumannii: A Metabolomic Study.XLSX
<p>Polymyxins are currently used as the last-resort antibiotics against multidrug-resistant Acinetobacter baumannii. As resistance to polymyxins emerges in A. baumannii with monotherapy, combination therapy is often the only remaining treatment option. A novel approach is to employ the combination of polymyxin B with non-antibiotic drugs. In the present study, we employed metabolomics to investigate the synergistic mechanism of polymyxin B in combination with the antineoplastic drug mitotane against polymyxin-susceptible and -resistant A. baumannii. The metabolomes of four A. baumannii strains were analyzed following treatment with polymyxin B, mitotane and the combination. Polymyxin B monotherapy induced significant perturbation in glycerophospholipid (GPL) metabolism and histidine degradation pathways in polymyxin-susceptible strains, and minimal perturbation in polymyxin-resistant strains. Mitotane monotherapy induced minimal perturbation in the polymyxin-susceptible strains, but caused significant perturbation in GPL metabolism, pentose phosphate pathway and histidine degradation in the LPS-deficient polymyxin-resistant strain (FADDI-AB065). The polymyxin B ā mitotane combination induced significant perturbation in all strains except the lipid A modified polymyxin-resistant FADDI-AB225 strain. For the polymyxin-susceptible strains, the combination therapy significantly perturbed GPL metabolism, pentose phosphate pathway, citric acid cycle, pyrimidine ribonucleotide biogenesis, guanine ribonucleotide biogenesis, and histidine degradation. Against FADDI-AB065, the combination significantly perturbed GPL metabolism, pentose phosphate pathway, citric acid cycle, and pyrimidine ribonucleotide biogenesis. Overall, these novel findings demonstrate that the disruption of the citric acid cycle and inhibition of nucleotide biogenesis are the key metabolic features associated with synergistic bacterial killing by the combination against polymyxin-susceptible and -resistant A. baumannii.</p
Table_2_Synergistic Killing of Polymyxin B in Combination With the Antineoplastic Drug Mitotane Against Polymyxin-Susceptible and -Resistant Acinetobacter baumannii: A Metabolomic Study.XLSX
<p>Polymyxins are currently used as the last-resort antibiotics against multidrug-resistant Acinetobacter baumannii. As resistance to polymyxins emerges in A. baumannii with monotherapy, combination therapy is often the only remaining treatment option. A novel approach is to employ the combination of polymyxin B with non-antibiotic drugs. In the present study, we employed metabolomics to investigate the synergistic mechanism of polymyxin B in combination with the antineoplastic drug mitotane against polymyxin-susceptible and -resistant A. baumannii. The metabolomes of four A. baumannii strains were analyzed following treatment with polymyxin B, mitotane and the combination. Polymyxin B monotherapy induced significant perturbation in glycerophospholipid (GPL) metabolism and histidine degradation pathways in polymyxin-susceptible strains, and minimal perturbation in polymyxin-resistant strains. Mitotane monotherapy induced minimal perturbation in the polymyxin-susceptible strains, but caused significant perturbation in GPL metabolism, pentose phosphate pathway and histidine degradation in the LPS-deficient polymyxin-resistant strain (FADDI-AB065). The polymyxin B ā mitotane combination induced significant perturbation in all strains except the lipid A modified polymyxin-resistant FADDI-AB225 strain. For the polymyxin-susceptible strains, the combination therapy significantly perturbed GPL metabolism, pentose phosphate pathway, citric acid cycle, pyrimidine ribonucleotide biogenesis, guanine ribonucleotide biogenesis, and histidine degradation. Against FADDI-AB065, the combination significantly perturbed GPL metabolism, pentose phosphate pathway, citric acid cycle, and pyrimidine ribonucleotide biogenesis. Overall, these novel findings demonstrate that the disruption of the citric acid cycle and inhibition of nucleotide biogenesis are the key metabolic features associated with synergistic bacterial killing by the combination against polymyxin-susceptible and -resistant A. baumannii.</p
Identification of Bzn-glutathione adducts.
<p>A. Upper plot is a chromatogram obtained from a sample of Bzn treated parasites corresponding to m/z 536.1922 within a 3 ppm mass range (single-charged ion). Middle and bottom plots are magnified mass spectra in the regions of interest within RT 18.5ā19. B. Chromatograms and m/z plots corresponding to m/z 268.5997 (di-charged ion) in a sample of Bzn treated parasites. In both A and B each ion is detected in two different but closely eluting chromatographic peaks with retention times centred at 17.8 and 18.6 minutes. Ionization charge states of the molecules are confirmed by the m/z difference for the isotopic <sup>13</sup>C peaks, which are expected to be 1.0034 for mono charged ions and 0.5017 for di-charged ions (bottom plots). <sup>34</sup>S isotopic peaks are also observed for both ions which are expected at 1.9959 m/z difference for mono-charged ions and 0.9979 for di-charged ions. C. MSMS fragmentation spectrum for precursor ion <i>m/z</i> 536.19. The m/z<sub>c</sub>ā=ā<i>m/z</i>ā1.007276 (proton mass). The proposed structures for the precursor ion (Bzn-glutathione adduct) and for some of the most intense fragments are shown. Exact theoretical mass and formula are included together with each fragment structure. A 4-C adduct is depicted though a 5-C adduct could be represented.</p
Evaluation of Coupling Reversed Phase, Aqueous Normal Phase, and Hydrophilic Interaction Liquid Chromatography with Orbitrap Mass Spectrometry for Metabolomic Studies of Human Urine
In this study, we assessed three liquid chromatographic
platforms:
reversed phase (RP), aqueous normal phase (ANP), and hydrophilic interaction
(HILIC) for the analysis of polar metabolite standard mixtures and
for their coverage of urinary metabolites. The two zwitterionic HILIC
columns showed high-quality chromatographic performance for metabolite
standards, improved separation for isomers, and the greatest coverage
of polar metabolites in urine. In contrast, on the reversed phase
column, most metabolites eluted very rapidly with little or no separation.
Using an Exactive Orbitrap mass spectrometer with a HILIC liquid chromatographic
platform, approximately 970 metabolite signals with repeatable peak
areas (relative standard deviation (RSD) ā¤ 25%) could be putatively
identified in human urine, by elemental composition assignment within
a 3 ppm mass error. The ability of the methodology for the verification
of nonmolecular ions, which arise from adduct formation, and the possibility
of distinguishing isomers could also be demonstrated. Careful examination
of the raw data and the use of masses for predicted metabolites produced
an extension of the metabolite list for human urine
Evaluation of Coupling Reversed Phase, Aqueous Normal Phase, and Hydrophilic Interaction Liquid Chromatography with Orbitrap Mass Spectrometry for Metabolomic Studies of Human Urine
In this study, we assessed three liquid chromatographic
platforms:
reversed phase (RP), aqueous normal phase (ANP), and hydrophilic interaction
(HILIC) for the analysis of polar metabolite standard mixtures and
for their coverage of urinary metabolites. The two zwitterionic HILIC
columns showed high-quality chromatographic performance for metabolite
standards, improved separation for isomers, and the greatest coverage
of polar metabolites in urine. In contrast, on the reversed phase
column, most metabolites eluted very rapidly with little or no separation.
Using an Exactive Orbitrap mass spectrometer with a HILIC liquid chromatographic
platform, approximately 970 metabolite signals with repeatable peak
areas (relative standard deviation (RSD) ā¤ 25%) could be putatively
identified in human urine, by elemental composition assignment within
a 3 ppm mass error. The ability of the methodology for the verification
of nonmolecular ions, which arise from adduct formation, and the possibility
of distinguishing isomers could also be demonstrated. Careful examination
of the raw data and the use of masses for predicted metabolites produced
an extension of the metabolite list for human urine
Evaluation of Coupling Reversed Phase, Aqueous Normal Phase, and Hydrophilic Interaction Liquid Chromatography with Orbitrap Mass Spectrometry for Metabolomic Studies of Human Urine
In this study, we assessed three liquid chromatographic
platforms:
reversed phase (RP), aqueous normal phase (ANP), and hydrophilic interaction
(HILIC) for the analysis of polar metabolite standard mixtures and
for their coverage of urinary metabolites. The two zwitterionic HILIC
columns showed high-quality chromatographic performance for metabolite
standards, improved separation for isomers, and the greatest coverage
of polar metabolites in urine. In contrast, on the reversed phase
column, most metabolites eluted very rapidly with little or no separation.
Using an Exactive Orbitrap mass spectrometer with a HILIC liquid chromatographic
platform, approximately 970 metabolite signals with repeatable peak
areas (relative standard deviation (RSD) ā¤ 25%) could be putatively
identified in human urine, by elemental composition assignment within
a 3 ppm mass error. The ability of the methodology for the verification
of nonmolecular ions, which arise from adduct formation, and the possibility
of distinguishing isomers could also be demonstrated. Careful examination
of the raw data and the use of masses for predicted metabolites produced
an extension of the metabolite list for human urine
Bzn <i>in vivo</i> derived metabolites arising after 20 ĀµM Bzn treatment of <i>T. cruzi</i> epimastigotes.
<p>Ions detected from 20 ĀµM Bzn treated parasites (cBt samples) and with low or no abundance in control samples (cBc, cTc, medium and solvent samples) are listed, retrieved from filtered or raw data. <b>RT</b>: retention time. <b>m/z<sub>c</sub>:</b> m/z values corrected for proton gain or loss (m/z<sub>c</sub>ā=āobserved <i>m/z</i> Ā± 1.007276). <b>IS</b>: Ionization state. 1, 2 and 3 refer to mono, di and tri charged ions respectively, from positive or negative ESI modes. Ionization was manually confirmed for all the listed metabolites, examining the m/z values of the <sup>13</sup>C related isotopic peaks. <b>Relative isotope abundance</b>: values from isotopic peaks were retrieved from filtered data or raw data. <b>Proposed formula</b>: most formulae were retrieved from BznMet database proposed metabolites. Some formulae were predicted using m/z data with IDEOM and rCDK <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002844#pntd.0002844-Guha1" target="_blank">[67]</a>. <b>Mass error (ppm):</b> [(m/z(observed)-m/z(exact))/m/z(exact)]*1E+6. <b>Mean cBt</b>: the mean peak intensity value for each ion in the corresponding study group (cBt). <b>Proposed metabolites</b>: proposed metabolites for each ion are listed with complete names or with short assigned names which include some selected features of the metabolites. der.: derivative. Complete IUPAC names and SMILES codes are included on <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002844#pntd.0002844.s002" target="_blank">File S1</a> āTargeted sheetā. All intensity values from raw data were retrieved manually (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002844#pntd.0002844.s004" target="_blank">File S3</a>). <b>Metabolite number:</b> numbers were assigned for cross referencing.</p><p>*<sup>34</sup>S isotopic peaks were not resolved from the <sup>13</sup>CII peaks in these metabolites.</p
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